DNA Repair

Interstrand crosslinks are cytotoxic lesions that inhibit essential processes including replication and transcription. Replication fork reversal occurs in response to interstrand crosslink inducing drug, MMC, but how replication fork reversal promotes repair of interstrand crosslinks is poorly understood. Here, we investigated the role of the RAD54L translocase in interstrand crosslink repair. We found RAD54L is required to promote nascent DNA degradation in FANCD2 and FANCA-depleted cells consistent with a previous study indicating RAD54L promotes replication fork reversal. We further show RAD54L activity is required for formation of radial chromosomes in FANCD2-deficient cells suggesting fork reversal may be required to generate the intermediate undergoing aberrant fusion in FANC-deficient cells. Finally, we demonstrate FANCD2 foci accumulate and DSBs persist in RAD54L-deficient cells indicating RAD54L is required for efficient repair of DSBs. Together, our results indicate RAD54L plays multiple roles in efficient processing and repair of interstrand crosslinks.

Caffeine, the most widely consumed stimulant worldwide and primarily sourced from coffee, is well known for its central nervous system effects. Emerging evidence indicates that caffeine also modulates key cellular processes, including DNA repair. It inhibits the kinase activity of ATM and ATR-essential DNA damage response proteins, and impairs homologous recombination (HR)-mediated repair through multiple mechanisms. However, its effects on nonhomologous end joining (NHEJ), a major double-strand break (DSB) repair pathway, have been underexplored. In a recent study, we reported that caffeine inhibits NHEJ primarily by interfering with Ligase IV/XRCC4 complex, using in vitro and ex vivo model systems. Given coffees role as a primary dietary caffeine source, this study investigates the impact of Coffea arabica decoction on NHEJ-mediated DSB repair. High-performance liquid chromatography (HPLC) quantified caffeine levels in the decoction, followed by in vitro and ex vivo assays to evaluate NHEJ efficiency. Results demonstrate that coffee decoction inhibits end joining of both compatible and noncompatible DNA ends in cell-free systems derived from normal and cancer cells. Extrachromosomal repair assays confirmed impaired intracellular NHEJ, leading to accumulation of unrepaired DSBs in human cells. Kinetic analysis of {gamma}-H2AX foci formation and resolution revealed persistent DNA breaks and reduced repair kinetics. Reconstitution experiments verified that the decoction specifically targets the Ligase IV/XRCC4 complex. These findings, building on our previous work, establish coffee decoction as a potent NHEJ inhibitor, mirroring purified caffeines effects. This underscores caffeines interference with endogenous DNA repair, with profound implications for cancer therapy by sensitizing tumors to genotoxic treatments.

Recent studies have suggested that S6 kinase 1 (S6K1) contributes to DNA repair (DR). However, the specific pathways and mechanisms involved in this regulation remain unclear. Moreover, it has not been investigated whether S6K2, a functional homologue of S6K1, also contributes to DR. In this study, we investigated the function of both S6K1 and S6K2 (S6K1/2) proteins in DR and demonstrate that both are important for efficient Homologous Recombination-mediated repair (HR). Double knockout of S6K1/2 prevented the formation of BRCA1 and RAD51 foci and increases sensitivity to DNA-damaging agents such as PARP1 inhibitors, cisplatin, and X-ray irradiation. In addition, double knockout of S6K1/2 increased markers of genomic instability, while single knockout had little effect on HR markers and genome stability, which suggests that one kinase can compensate for the loss of the other. Mechanistically, we show that S6K1/2 regulate BRCA1 protein stability, limiting its degradation by the proteasome. Finally, pharmacological inhibition of S6K1/2 sensitised HR-proficient breast cancer cells to Olaparib. Our findings clarify the role of S6K1/2 proteins in HR and suggest that targeting these kinases may be a therapeutic strategy to enhance PARP inhibitor efficacy in HR-proficient tumours.

Ribonucleotides are frequently incorporated into our genome during replication. Canonically, RNase H2 is responsible for the removal of these embedded ribonucleotides. Alternatively, DNA topoisomerase 1 (TOP1) has also been shown to have genomic ribonucleotide processing activity. When this process occurs at short tandem repeat (STR) sequences, it can lead to 2-5 bp deletions. These deletions are the result of two sequential cuts by TOP1 at sites of ribonucleotide incorporation. In this study, we have determined that PARP1 regulates the TOP1-mediated excision of ribonucleotides by preventing the formation of TOP1-DNA adducts that occur through a second cleavage following the initial ribonucleotide cut by TOP1. We biochemically defined the mechanism by which this regulatory inhibition of TOP1 occurs, which involves both PARP1 physically restricting TOP1 from the cleavage site followed by the inhibitory PARylation of TOP1. We also show that this activity means that PARP1 prevents the TOP1-dependent deletions at STRs in cells. In the absence of both a functional RNase H2 complex and PARP1, we demonstrated that cells appear to be in a senescent state provoked by the accumulation of TOP1-DNA adducts, which are a result of TOP1 being unimpeded to remove genomic ribonucleotides. Our work has elucidated the role of PARP1 in preventing the deleterious consequences of the processing of genomic ribonucleotides by TOP1. Understanding this mechanism could help us develop therapies that better sensitize tumors to PARP inhibitors, especially in cancers that present loss-of-function RNase H2 mutations (seen in certain chronic lymphocytic leukemia and prostate cancers).

B-cell leukemia/lymphoma 11B (BCL11B), despite its name, is a key regulator of T-cell development, specification, and T-cell malignancies. BCL11B contains a bipartite DNA binding domain composed of two C2H2 zinc finger arrays: low-affinity ZF2-3 and high affinity ZF4-6. These arrays function as homotypic modules that recognize similar six-nucleotide motifs, TG(O_SCPLOWNC_SCPLOW)CC(O_SCPLOWCC_SCPLOWO_SCPCAP/C_SCPCAPO_SCPLOWTC_SCPLOWO_SCPCAP/C_SCPCAPO_SCPLOWAC_SCPLOW), as seven of the eight DNA base-contacting residues are conserved between them. The most conserved interactions involve GG dinucleotides, contacted by arginine and lysine residues at key base-interacting positions in ZF3 and ZF5. The two ZF arrays are connected by a long [~]300-residue linker that provides flexibility in how the arrays engage DNA, allowing ZF2-3 and ZF4-6 binding to the same or opposite strands with variable orientation, spacing and positioning along the DNA. This extended linker is enriched in serine/threonine, acidic residues (aspartate/glutamate), and structural residues (glycine/proline), providing additional layers of transcriptional regulation possibly through post-translational modification, electrostatic modulation, and/or condensate formation. We also examined six missense mutations in base-interacting residues, that are associated with neurodevelopmental disorders. Substitutions replacing bulky, positively charged arginine or lysine with smaller or hydrophobic residues likely reduce DNA-binding affinity and/or specificity, whereas substitutions between asparagine and lysine may alter base recognition preferences.

Latent HIV-1 proviruses remain the major barrier to curing HIV infection. Although many of these proviruses are defective, with large internal deletions and hypermutations, the mechanisms underlying their formation are still poorly understood. In this study, we applied CRISPR/Cas9 knockout screens to identify DNA damage response (DDR) proteins that contribute to the formation of defective HIV-1 proviruses carrying large internal deletions. Using an HIV-1-based dual-fluorophore vector as a model, we distinguished cells harbouring intact proviruses from those carrying large internal deletions by flow cytometry and cell sorting. We then validated top candidates using CRISPR-mediated gene activation and small interfering RNA-mediated knockdown, and we measured gene and protein expression by quantitative PCR and Western blotting. Across these approaches, the helicase-like transcription factor HLTF emerged as a consistent modulator of large internal deletions: increased HLTF expression raised the proportion of cells carrying defective proviruses, whereas reduced HLTF expression had the opposite effect. Additional repair factors, including RAD1, RAD18, TREX2, and ZRANB3, also influenced the balance between intact and defective proviruses, suggesting that multiple DNA repair pathways cooperate in this process. Deep sequencing of reporter proviruses confirmed the presence of large internal deletions in the populations identified as defective. Our data indicate that several DNA damage response proteins, including HLTF, are involved in the generation of defective proviruses and may constitute a previously undescribed host defense mechanism against HIV-1. Authors SummaryWhen HIV-1 infects a cell, it copies its genetic material (RNA) into DNA and inserts this DNA into the cells genome, giving rise to proviruses that can persist for long periods and become part of the host DNA. Many of these viral DNA copies are defective, often missing large parts of their genome, but we still do not fully understand how these large deletions arise. In this study, we used a genetic screening approach to switch off many human DNA repair genes and asked how this affected the balance between intact and defective HIV proviral DNA. We used an HIV-1-based dual-colour reporter vector allowing us to distinguish intact from deleted viral DNA by simple fluorescence read-outs. We found that several human DNA repair factors, in particular a protein called HLTF, change how often large deletions appear. Our results suggest that normal DNA repair processes in infected cells can sometimes turn incoming HIV-1 DNA into defective forms that cannot support productive infection. This work points to host DNA repair as a contributor to the large pool of defective HIV-1 DNA seen in people with HIV (PWH) and raises the possibility that these pathways could one day be harnessed to make infections less harmful.

The MYC associated factor X (MAX) is the heterodimeric partner of the MYC paralogs (MYC, MYCN and MYCL). When deregulated, high level of the MYC paralogs contribute to all aspects of tumorigenesis and tumor growth. MAX can also heterodimerize with the MXD proteins, MNT and MGA. Heterodimerization and sequence specific DNA binding to the E-Box sequences at gene promoters is controlled by their heterodimerization with the MAX b-HLH-LZ. As a heterodimer with MAX, MYC proteins activate genes involved in cell metabolism, growth and proliferation whereas MXD proteins, MNT and MGA repress them. MAX can also bind to the E-Bos sequence as a homodimer. Being devoid of a transactivation domain it can act as an antagonist of the MYC/MAX heterodimers. Variants of MAX have been reported to be linked to cancer. These variants are either not expressed, inactivated or lead to missense mutations. This has led to the notion that MAX may have a tumor suppressor role. Here, we characterize three of those variants with missense mutations in the basic region, i.e. E32K, R35P and R35C. We analyzed their heterodimerization with the b-HLH-LZ of MYC and their DNA binding properties as homo-and heterodimers. The R35C variant b-HLH-LZ was found to have a markedly increased affinity for the b-HLH-LZ of MYC. We also observed that all three b-HLH-LZ variants have a lower affinity as homodimers for the E-Box than the WT. This was shown to lead to a preferential binding of all the heterodimeric b-LHLH-LZ to the E-Box. This effect is exacerbated in the case of the R35C variant. We argue that this preferential binding of MYC as heterodimers with these variants to E-Box sequences could contribute to tumorigenesis. Hence, our results suggest that, mechanistically, the MAX homodimer bound to the E-Box could act as a tumor suppressor. MATERIALS AND METHODSO_ST_ABSMolecular modelingC_ST_ABSThe open source version 1.7.6.0 of Pymol was used for modeling and molecular rendering [1]. The crystal structure of the MAX homodimer bound to the E-Box (1HLO [2]) was used as a template for the generation of the models. The variants were generated using the mutagenesis function in the wizard. The conformation of the K32 side chain was manually set in order to avoid introducing steric clashes with DNA. Protein expression and purificationThe cDNA, coding for the MAX b-HLH-LZ (Max* hereafter, residues 22-103, UniProt entry P61244-1) to which are added the GSGC residues in c-terminal, inserted in the pET3a vector was already available in the laboratory [3] and was used as a template to generate the plasmids with inserts coding for each of the mutants (E32K, R35C and R35P) through quick-change PCR with Q5 DNA polymerase and DpnI from New England Biolabs. The primers used were purchased from IDT DNA, their sequences are listed in Table S1. Sequence for each construct was confirmed by Sanger sequencing at the Plateforme de sequencage SANGER - Centre de recherche du CHU de Quebec - Universite Laval. The primary structure for the basic region of each construct is given in Fig. 2A. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/715400v1_fig2.gif" ALT="Figure 2"> View larger version (41K): org.highwire.dtl.DTLVardef@1b05d5eorg.highwire.dtl.DTLVardef@1c1d692org.highwire.dtl.DTLVardef@ee469dorg.highwire.dtl.DTLVardef@15e0ba4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 2.C_FLOATNO Structure schematics, specific and non-specific interactions dictating specificity and stability of binding of the basic region of MAX to the canonical (CACGTG) E-Box. A. Primary structure for the basic region of MAX and each of the variants. Positions making the most important contacts with the E-box are indicated by black arrows. Positions for the variants studied here are colored according to the Zappo colour scheme, following their physico-chemical properties: red for negative, blue for positive, magenta for proline and yellow for cysteine. B. The side chain (carboxylate) of E32 receives H-Bonds from the CA nucleobases in the leading strand (white carbon atoms). R35 and R36 make a salt bridges with phosphate groups while and the guanidino moiety of R36 makes a specific H-Bond with the nucleobase of the G in the strand of the reverse complement (cyan carbon atoms). C. The R35C mutation removes one non-specific salt-bridge at the interface of the complex. D. The aliphatic portion of the K side chain in the E32K variant is unable to accept the H-Bonds from the CA nucleobases and leads to the stabilisation of the complex and the helical structure of the basic region. E. In addition to removing a salt-bride, the Pro residue in the R35P kinks the path of the basic region, prevents the establishment of the specific H-Bonds mandatory for recognition of the E-Box and leads to unfolding of the helical state. C_FIG The MYC b-HLH-LZ (Myc*), the Max*WT b-HLH-LZ and its variants were expressed and purified as previously described [3,4] After lyophilisation, the b-HLH-LZs were kept at -20{degrees}C and solubilised in Myc buffer (50 mM NaCl, 50 mM NaH2PO4 pH 5.5) for Myc* or PBS for Max* at a final concentration of 1 mM before use. Circular dichroismAll circular dichroism (CD) measurements were performed on a Jasco J-810 spectropolarimeter equipped with a Peltier-type thermostat. The instrument was routinely calibrated using an aqueous solution of d-10-(+)-camphorsulfonic acid at 290.5 nm. Samples were prepared as follows: Max* (either WT or a variant) was diluted in 100 {micro}l 2X CD buffer (40 mM KCl, 11.4 mM K2HPO4, 28.6 mM KH2PO4, pH 6.8) and the volume adjusted to 106 {micro}l with PBS. 10 {micro}l TCEP 16 mM were added, and the volume further adjusted to 192 {micro}l with ddH2O before samples were incubated overnight at room temperature. After reduction, Myc* was added and the volume adjusted to 198 {micro}l with Myc buffer (Na2HPO4 0.95 mM, NaH2PO4 49.05 mM, 50 mM NaCl, pH 5.5). The DNA complexes were prepared as follows. After a 10 minutes incubation of the protein samples at room temperature, 0, 1 or 2 {micro}l of 2 mM of specific or non-specific DNA duplexes in 10 mM Tris pH 8.0 were added and the volume adjusted to 200 {micro}l with 10 mM Tris pH 8.0. The strands of the specific probe were: 5-ATT ACC CAC GTG TCC T*AC-3 and 5-GTA GGA CAC GTG GGT* AAT-3 (with the E-box sequence underlined) and the non-specific probe: 5-ATT ACC TCC GGA TCC T*AC-3 and 5-GTA GGA TCC GGA GGT* AAT-3 (Integrated DNA Technologies). Samples were further incubated for 10 minutes at room temperature and transferred to a 1 mm path length quartz cuvette. All spectra were recorded from 250 to 195 nm at 0.1 nm intervals by accumulating 10 spectra at 25 {degrees}C. Thermal denaturations were recorded at 222 nm from 5 to 95 {degrees}C at a heating rate of 1 {degrees}C/min. CD signal for spectra and thermal denaturations was corrected by substracting the signal from corresponding spectra or thermal denaturation either for buffer alone or the appropriate DNA duplex. CD signal was then converted to mean residue ellipticity using the following formula [5]: [{theta}] = {delta} {middle dot} MRW/(10{middle dot}c l) where [{theta}] is the mean residue ellipticity in deg {middle dot} cm2 dmol-1, {delta} is the CD signal in millidegrees, MRW is the mean residue weight, c is the concentration in mg/ml and l is the pathlength in mm. For the heterodimers, the concentration used was the sum of Max* and Myc* and the MRW was determined using a weighted average.

Formaldehyde (FA) is an environmentally abundant and endogenously produced aldehyde that has been shown to cause DNA damage, mutagenesis, and carcinogenesis. Several studies have demonstrated that FA induces guanine mutations resulting in a mutation signature like SBS40. In this work, we demonstrate that replication defects generating single-stranded DNA (ssDNA) caused by the downregulation of the major replicative polymerases results in elevated FA mutagenesis. We found that loss of Mrc1 (CLASPIN) resulted in a high accumulation of ssDNA and FA mutagenesis, and that these phenotypes were not dependent on Mrc1s checkpoint activity. Loss of DNA-protein crosslink repair results in elevated FA sensitivity with no alteration to mutagenesis, likely due to the inability of the fork to bypass unprocessed protein adducts. Finally, we show that FA-induced mutagenesis is dependent on Pol {zeta}-mediated translesion synthesis, while deficiencies in the template switching pathway do not alter error-free bypass of FA adducts. Overall, our work points towards replication-associated ssDNA as a major substrate for FA-induced damage and elucidates the pathways that function to prevent FA mutagenesis at replication forks.

BackgroundDynamic chromatin behavior, which is related to chromatin accessibility, plays a critical role in various genome DNA functions such as RNA transcription and DNA replication/repair. Previous studies using highly synchronized cells showed that average local chromatin motion, captured by single-nucleosome imaging and tracking on a second time scale, remained almost constant throughout G1, S, and G2 phases in living human cells, although possible effects of prolonged drug treatments for cell-cycle synchronization could not be excluded. ResultsTo avoid possible effects of prolonged drug treatment, we combined single-nucleosome imaging with Fucci probes to visualize cell-cycle progression through G1, S, and G2. Using HeLa and HCT116 cells expressing H2B-HaloTag and Fucci probes, we found that local nucleosome motion remained similar on average throughout interphase, except for elevated motion in early G1. Transcription inhibition similarly increased nucleosome motion throughout interphase. Local nucleosome motion also increased following replication stress or DNA damage. ConclusionOur findings suggest that near-constant chromatin motion supports housekeeping functions under similar physical conditions during interphase. Our findings also suggest that cells can transiently change chromatin motion to perform ad hoc tasks in response to signals from inside and outside the cell, such as DNA damage.

Telomeres are nucleoprotein structures at the ends of eukaryotic chromosomes that safeguard them from triggering inappropriate DNA damage signaling. POT1, a member of the mammalian shelterin complex, binds single-stranded (ss) telomeric DNA and blocks the activation of the ATR kinase-mediated DNA damage response at telomeres. Yet until recently, it was poorly understood how the double-stranded (ds)-ss telomeric junction was protected from DNA damage response factors. An initial study of the DNA-binding activity of human POT1 (hPOT1) using systematic evolution of ligands by exponential enrichment (SELEX) and subsequent investigation revealed that POT1 contains a binding pocket, known as the POT-hole, that binds the 5 phosphorylated dC of the telomeric ds-ss junction. The amino acid residues composing the POT-hole show full sequence identity with telomeric proteins from diverse eukaryotes, including Caenorhabditis elegans POT-1. The current study builds on this SELEX method, developing an extensive analysis pipeline for SELEX datasets sequenced by next-generation sequencing and achieving a deeper analysis of the resulting sequences. We validated our approach by applying it to the DNA-binding domain of hPOT1, yielding results consistent with a previous SELEX study. Furthermore, we employ our pipeline to characterize the DNA-binding activity of C. elegans proteins that are considered homologs of hPOT1: POT-1, POT-2, POT-3, and MRT-1. Our analysis suggests that all four proteins show a binding preference for G-enriched DNA sequences, with POT-1 additionally binding secondary structural elements. Overall, we present a bioinformatics pipeline that is accessible and applicable for determining the nucleic acid-binding properties of a variety of proteins.

DNA-binding proteins must quickly locate specific sites on DNA to enable replication, repair, and transcription. While sequence-specific recognition is well understood, the physical basis of structure-specific recognition remains unclear, limiting our understanding of DNA damage repair. Proteins must distinguish damaged sites within largely undamaged DNA; however, studying this is challenging due to DNAs dynamic nature. We hypothesised that DNA damage causes changes in DNA structure, signalling protein recruitment. Using confocal single-molecule FRET, we analysed seven DNA duplexes containing modifications such as ribonucleotide, 8-oxoguanine (8-oxoG), abasic sites, nicks, and gaps, which are all involved in the base excision repair (BER) pathway. Each construct was measured with nine dye pairs in triplicate to capture changes in bending, twisting, and stretching. An automated analysis pipeline processed 162 measurements, enabling rigorous statistical comparisons. All modifications altered FRET efficiencies compared to undamaged DNA, including the subtlest change: a single oxygen difference (ribo-vs deoxyribonucleotide). Abasic sites, nicks, and gaps had the greatest effects. These findings provide direct evidence that DNA damage affects duplex structure and dynamics beyond the lesion site, suggesting DNA flexibility changes may act as an early signal for repair protein recruitment. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/709887v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@a85839org.highwire.dtl.DTLVardef@3813dborg.highwire.dtl.DTLVardef@19fa06aorg.highwire.dtl.DTLVardef@dc9729_HPS_FORMAT_FIGEXP M_FIG C_FIG

Anaplastic thyroid carcinoma (ATC) is one of the most lethal malignancies. Immune dysregulation is believed to play an important role in ATC. Here, we aimed to characterize the systemic inflammation and the function of circulating immune cells of patients with ATC. First, we retrospectively assessed biochemical parameters of patients with ATC and observed that high systemic inflammation correlated with worse survival. Next, we prospectively investigated the inflammatory proteome, single-cell peripheral blood mononuclear cell transcriptome and epigenetic changes. Circulating concentrations of proinflammatory cytokines were increased in ATC patients. This proinflammatory profile was apparent at the level of gene transcription and chromatin accessibility, especially in monocytes. These findings were substantiated by an increased capacity of peripheral blood mononuclear cells of ATC patients to produce IL-6, IL-8 and lactate. As IL-6 is known to promote tumor cell survival, we assessed its capacity to influence ATC cell proliferation. Blocking IL-6/gp130/Jak/STAT3 pathway inhibited proliferation of ATC cell lines in vitro. In conclusion, these findings show that ATC is characterized by inappropriate systemic inflammation and epigenetic and transcriptional reprogramming of circulating monocytes. Proinflammatory cytokines released by monocytes support survival and proliferation of ATC tumor cells, suggesting a therapeutic potential of targeting this pathway in ATC patients.

The repair of photo-induced DNA lesions through nucleotide excision repair machinery is still the source of important questions. It has been observed that the repair rate of the different cyclobutane pyrimidine dimers, i.e. the photoproducts induced by dimerization of two {pi}-stacked pyrimidines (T<>T, T<>C, C<>T, C<>C), depends on the nucleobases involved in the lesion. TT derivatives (T<>T) are removed more slowly than those containing cytosine, especially in 5. Using all-atom molecular dynamics simulations and free-energy calculations, we demonstrate that the variation of the repair rate observed in human skin and in cultured cutaneous cell is associated to the recognition of the four lesions by the DDB2 protein moiety, and more specifically by the differential structural deformation induced on the complementary strand. Indeed, while C<>C and C<>T induce a larger deviation on the groove parameters, T<>T and T<>C, instead, affect DNA structure to a lesser extent. less affected. These effects then hamper differentially the downstream recruitment of the repair complexes. The observed DNA deformation correlates with the experimental repair rate and provides a structural rationale for the different repair rates of CPD by nucleotide excision repair machinery. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/724087v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@cf6b6dorg.highwire.dtl.DTLVardef@195e35forg.highwire.dtl.DTLVardef@1829296org.highwire.dtl.DTLVardef@165baba_HPS_FORMAT_FIGEXP M_FIG C_FIG

Bdelloid rotifers are known to survive desiccation and high doses of ionizing radiation. This extreme resistance is notably due to their capacity to cope with numerous DNA double-strand breaks (DSBs). Genes encoding key components of the non-homologous end joining (NHEJ) DNA repair pathway are strongly upregulated in the bdelloid rotifer Adineta vaga following exposure to ionizing radiation. Considering the notably high doses tolerated by these organisms, their capacity to efficiently restore genome integrity is particularly striking. Although NHEJ is generally regarded as less accurate than homologous recombination (HR), the absence of major genomic rearrangements in the descendants of irradiated rotifers suggests that DNA repair occurs with high fidelity. Terwagne et al. recently reported a delayed repair in germline nuclei, occurring during oocyte development when homologous chromosomes pair, thereby enabling template-based repair through HR. In this study, we established an in situ hybridization approach on A. vaga cryosections to investigate the spatial and temporal expression of key actors involved in NHEJ, HR, and Base excision repair (BER) pathways in somatic and germline tissues. We show that NHEJ (KU80) and BER-related genes (PARPs) as well as A. vaga Ligase E (putatively involved in DNA repair) are expressed early after radiation exposure in the somatic syncytium. In contrast, HR-related genes (Rad51: two paralogs, Rad54), as well as PCNA (involved in DNA replication, NER, BER, HR) are expressed later in maturing oocytes, indicating the activation of a delayed homologous recombination repair pathway in germline nuclei. Nurse cells, which express genes associated with both HR and NHEJ pathways, may rely on both mechanisms for their own DNA repair while also supplying mRNAs to the maturing oocyte. Our results provide new evidence for a differential regulation of DNA DSB repair pathways between soma and germline in bdelloids, with NHEJ predominating in somatic tissues and HR in the germline of A. vaga. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/722046v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@3b1f3borg.highwire.dtl.DTLVardef@17f5eb5org.highwire.dtl.DTLVardef@122ef14org.highwire.dtl.DTLVardef@7e4413_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOAbstract Figure:C_FLOATNO Summary of in situ hybridization results: genes coding for actors of NHEJ are expressed in the somatic nuclei and in the nurse nuclei of Adineta vaga individuals 2.5 hours post X-rays radiation, while genes coding for HR actors and PCNA (involved in multiple pathways including DNA replication and DNA repair: NER, BER, MR, HR) are expressed in the nurse nuclei 2.5 hours post radiation, and later in the maturing oocyte during oogenesis and in the laid eggs. Genes coding for actors highly expressed post-radiation, involved in the BER pathway appear to be only expressed in the somatic syncytium 2.5 hours post radiation, as well as the gene coding for the Ligase E, likely involved in DNA repair. C_FIG

One challenge to DNA replication is the presence of unrepaired damage on the template strand, which can stall the replication machinery. This stall can be resolved by the translesion synthesis (TLS) pathway, in which specialized translesion polymerases are recruited to copy damaged DNA. Because TLS polymerases are error-prone, their activity is regulated at multiple levels to minimize unnecessary mutagenesis. Although the molecular mechanisms of bacterial TLS have been extensively studied in Escherichia coli, less is known about this pathway in other species. In E. coli, the TLS polymerase Pol IV is minimally enriched at replication forks in the absence of DNA damage but is strongly recruited upon replication stalling, enabling TLS while minimizing mutagenesis. However, we recently showed that the Bacillus subtilis TLS polymerase Pol Y1, the homolog of Pol IV, is moderately enriched near replication sites even during normal growth and is not further enriched upon treatment with the DNA damaging agent 4-nitroquinoline 1-oxide (4-NQO). It is unknown whether this behavior is unique to 4-NQO or general to other types of DNA damage. In this study, we investigate the effects of four different DNA damaging agents (ultraviolet light, methyl methanesulfonate, nitrofurazone, and mitomycin C) in B. subtilis. We first characterize the contributions of the two TLS polymerases, Pol Y1 and Pol Y2, to DNA damage survival and damage-induced mutagenesis after treatment with these agents. We then use single-molecule fluorescence microscopy to measure the localization and dynamics of individual Pol Y1 molecules in live B. subtilis cells. We find that Pol Y1 and Pol Y2 have differing effects on survival and mutagenesis, but that under no circumstances is Pol Y1 strongly recruited to sites of replication upon DNA damage. This study broadens our understanding of TLS in B. subtilis, indicating that there are notable differences in TLS mechanisms across bacteria.

PurposeLow-dose radiation-induced adaptive response (LDRIAR) is well documented, but its role in early DNA damage signalling remains unclear. This study aimed to investigate whether adaptive response influences initial DNA double-strand break (DSB) recognition, as reflected by {gamma}H2AX foci formation, and to evaluate its time-dependent expression in human lymphocytes. Materials and MethodsPeripheral blood lymphocytes from three healthy donors were exposed to a priming dose followed by a challenging dose at defined time intervals. DNA damage was assessed using {gamma}H2AX foci analysis, comparing acute and split-dose exposures in both PHA-stimulated (large) and non-stimulated (small) lymphocytes. ResultsA clear time-dependent adaptive response was observed. No significant reduction in {gamma}H2AX foci was detected at 1 h (p > 0.05). At 2 h, a significant decrease was observed ([~]7-8% in large and [~]13% in small lymphocytes; p < 0.01), which increased at 4 h ([~]12% and [~]22%, respectively; p < 0.001). The maximal response occurred at 15 h, with reductions of [~]40- 43% in large and [~]27% in small lymphocytes (p < 0.001). Small lymphocytes exhibited an earlier response, while large lymphocytes showed a greater magnitude at later time points. The temporal trend was consistent across donors, with minor variability at later intervals. ConclusionsThe findings demonstrate that LDRIAR is reflected at the level of DNA damage signalling and follows a defined temporal pattern with cell-type specificity. This suggests that adaptive response may influence early DSB-associated processes, contributing to a better understanding of radiation response mechanisms in radiobiology.

Type III restriction-modification (RM) enzymes are prominent bacterial defense against bacteriophage and invading foreign DNA that also modulate the hosts epigenetic landscape. Genome analysis of the host-adapted Mycoplasma bovis PG45 that has a very small genome revealed a Type III RM locus comprising one res and three mod genes. We characterized Mbo45V, a representative enzyme encoded by this locus. The enzyme forms a heterotrimeric complex consisting of two Mod subunits and one Res subunit. Mbo45V recognizes the asymmetric sequence 5'-YAATC-3' (Y = T/C) and cleaves DNA having at least two head-to-head oriented sites [~]26-28 bp away from the recognition site. Methylation of the second adenine of the target site using cofactor S-adenosylmethionine (SAM) protects DNA from restriction, while the SAM analogue sinefungin enhances DNA binding and cleavage. Kinetic studies reveal that Mbo45V exhibits relatively weak DNA binding affinity and an unusually high Km for SAM, indicating low cofactor affinity compared to prototypical enzymes such as EcoP15I. ATPase activity is strongly stimulated by cognate DNA and is inhibited upon methylation of the substrate, suggesting a regulatory interplay between methylation and restriction functions. Comparative analysis indicates that, although Mbo45V shares core mechanistic features with prototypes from Escherichia coli, its kinetic parameters are distinct. These differences likely reflect adaptation to the stable intracellular environment of M. bovis, in contrast to the fluctuating conditions encountered by the enteric bacteria.

DNA helicases are ATP-dependent motor proteins that catalyze duplex DNA unwinding and are involved in DNA repair, recombination and replication restart. Prominent members of the non-hexameric SF1A UvrD-family helicases are E. coli UvrD, Rep, B. stearothermophilus PcrA and M. tuberculosis UvrD1. SF1A monomers are processive 3 to 5 single stranded DNA translocases, but need to be activated to become DNA helicases. One mechanism of activation is dimerization. Whereas Rep, UvrD and PcrA form non-covalent dimers, the Mtb UvrD1 helicase forms a redox-dependent covalent dimer. Dimerization of Mtb UvrD1 occurs between the same regulatory domain (2B) within each subunit stabilized by a disulfide bond formed between the same cysteine (Cys451) within each subunit. Dimerization relieves an inhibitory interaction between the 2B domain and duplex DNA within the monomer-DNA complex. We show here that Rep, UvrD and PcrA dimerize using the same 2B-2B interface. By placing a Cys residue within the 2B domains of Rep, UvrD and PcrA in the structurally equivalent position occupied by Cys451 of Mtb UvrD1, all three enzymes form redox-dependent covalent dimers that are constitutively active helicases with increased processivity compared to the non-covalent dimers. Hence, the 2B domain is a general dimerization domain for UvrD-family SF1A helicases.

The cellular response to DNA replication stress (DRS) provoked by anticancer drugs involves activation of the G2/M checkpoint (which promotes transient cell cycle arrest at G2 phase) and DNA repair, followed by induction of apoptosis or senescence. Here, we activated the p53-p21 pathway and ATR using DRS-inducing drugs, and found that that the transition to senescence depends on the duration of the G2 phase. Shortening of G2 duration by G2/M checkpoint inhibitors led not only to a switch in cell fate from senescence to mitotic entry, but also to effective cell death through carry-over of chromosomal aberrations (generated by DRS-inducing drugs) into mitosis and subsequent mitotic progression. Such enhanced cell death was also observed in p53 deficient cells, which do not normally undergo senescence. Thus, we propose that temporal regulation of G2 phase is an approach to enhancing the effects of DRS-inducing drugs in a manner that is independent of p53 status.

Escherichia coli YicC enzyme is the founding member of a family of endoribonucleases that is encoded in virtually all bacterial species. Previous structural studies revealed that this ribonuclease binds RNA by a novel mechanism in which the hexameric apoprotein presents an open channel that undergoes a large rotation upon RNA binding and clamps down on the RNA. The current study follows up on these findings by examining the cleavage of various oligonucleotide substrates designed to probe recognition elements required for YicC binding and cleavage. A 26-nucleotide RNA oligomer (oligo), with a KD in the low micromolar range, was the standard to which numerous oligos with altered sequence were compared. In vitro RNase assays and fluorescence anisotropy binding measurements indicated that the preferred substrates for YicC were relatively small RNAs that contain some secondary structure. Larger RNAs or highly structured RNAs were less-than-optimal substrates. Similarly, RyhB RNA, a [~]90-nucleotide, iron-responsive RNA of E. coli, which has been described as a target of YicC binding and/or cleavage, was a poor YicC substrate in our assays. These results suggest that the native substrates for YicC-family members are very small RNAs or RNA fragments derived from larger RNAs.